Cyclophosphamide and human renal transplantation. e.W. PUTNAM, M.D., B. LAUNOIS, M.D., 1. CORMAN, M.D.,. G. SCHROTER, M.D.. A. S. BOOTH, Jr., M.D. 1 ...
Cyclophosphamide and human renal transplantation M.D., B. LAUNOIS, M.D., 1. CORMAN, M.D., G. SCHROTER, M.D. A. S. BOOTH, Jr., M.D. 1. PENN, M.D. C. G. HALGRIMSON, M.D. e. G. GROTH, M.D. T. E. STARZL, M.D., Ph. D.
e.W. PUTNAM,
From the Departments of Surgery and Pediatrics, University of Colorado School of Medicine and the Veterans Administration Hospital Denver. Colorado, U.S.A.
In this discussion we propose to describe the use of cyclophosphamide in immunosuppressive regimens that recently have been employed for humans after whole organ transplantation. Before doing this it might be well to briefly go back over some major principles of immunosuppressive therapy that were worked out almost a decade ago with drugs other than cyclophosphamide. Almost all of this basic information came from the accurate observation of patients subjected to the relatively simple operation of renal homotransplantation. REVERSAL
OF
REJECT/ON AND « ACCEPTANCE»
RELATIVE
GRAFT
Some typical events subsequent to renal homotransplantation are illustrated in Figure I. This 23-year-old patient was given a renal homograft by his younger brother in early 1963. After transplantation, the drug azathioprine (Imuran R ) was given in an attempt to prevent rejection. There was prompt and excellent function of the new kidney with large volumes of urine, a spectacular rise in creatinine clearance, and a fall in BUN. However, after a little more than 2 post-operative weeks the new kidney began to fail. The creatinine clearance dropped sharply, the BUN rose secondarily, the urine volume diminished and the patient gained weight. In addition, there was an increase in the white cell count. He developed hypertension which required control by antihypertensive medications,
* This work was supported by research grants from the Veterans Administration, by grants RR-OOOSJ and RR-00069 from the general clinical research centers program of the Division of Research Resources, National Institutes of Health, and by grants AI10176-01, AI-AM-08898, AM-077n, and HE-09JJO of the United States Public Health Service. 35
C.W. Putnam et al. PATIENT J. R. (AGE 23 if)
URINE OUTPUT (cc/day)
CCr (cc/mln)
BUN (mgm%)
IMURAN (mgm/day)
TIME IN DAYS
Fig. l. - Classical rejection crisis III a patient being treated with the doubledrug combination of azathioprine (lmuran) and prednisone. Deterioration of renal function b~gan 17 days after transplantation. All stigmata of rejection were present except for acute hypertension and weight gain, which were successfully prevented by medical treatment. The patient, whose transplantation was on April 17, 1963, still has excellent function of the same homograft 9 years later. Biopsy of the homograft after two years was normal. Acti-C - actinomycin C; LN left nephrectomy at the time of transplantation; RN - right nephrectomy. (By permission of Surg. Gynec. Obstet. 1 J 7: 385, 1963).
became acutely febrile with temperatures of almost 40 'C, and developed proteinuria. In short, all the manifestations of acute rejection had developed in spite of immunosuppressive therapy, namely signs of acute homograft failure plus an acute systemic febrile illness. Before experience with this patient and others of the same era in the early 1960's, it had been assumed that rejection was one of biology's most vigorous and persevering reactions and that, once begun, it would continue to the death of the transplant. It was now proved that this assumption was false and that rejection was a highly reversible process. In this case, immunosuppressive treatment was intensified by the addition of prednisone to the azathioprine. The findings of rejection promptly disappeared. The BUN fell, the creatinine clearance rose, and the proteinuria disappeared as did the high blood pressure. The fever was gone within a few hours of the institution of the prednisone therapy. Rejection had thus been shown to be a reversible process. This was a fundamental disclosure by clinicians and one 36
Cyclophosphamide and human renal transplantation which had not been at all anticipated by previous workers in basic research laboratories. The second equally important thing learned from these early patients can also be demonstrated by the same case. In the weeks after rejection had been reversed in this patient, the prednisone dose was very rapidly reduced, and eventually it was discontinued altogether after only about 5 months. Then the azathioprine dose was also lowered until within a year the patient was receiving only about half the daily quantities that at the outset had failed to prevent the onset of a moderately severe rejection. It is quite possible that all immunosuppressive therapy could be stopped in this patient who is still alive with perfect renal function after almost 9 years. Although we have ·never felt justified in taking the final drastic step of stopping treatment in any human recipient, we have thoroughly tested the hypothesis in dogs. We have animals in our laboratory surviving for as long as 9 years and 8 years, respectively, after renal and hepatic homotransplantation from non-related mongrel donors. These remarkable dogs who, it is now clear, are apt to die of old age, were treated with azathioprine for only a few months post-operatively and have not now received any treatment for approximately 2 / 3 of a canine lifetime. Even in this introduction it is worth summarizing the two concepts which have just been stated. First, rejection is a highly reversible process. Second, a favorable change often occurs after transplantation by virtue of which the host comes to better tolerate the presence of the homograft, thereby allowing immunosuppression to be reduced. If either of these statements were not true, organ transplantation would not be feasible. The fact that they are true is the inside story of clinical transplantation. The observations supporting these two conclusions indicate that whole organ homotransplantation in conjunction with non-specific immunosuppressive therapy can and often does lead to selective abrogation of the host rejection response. From a practical point of view, this objective is most easily achieved by the use of pharmacologie agents in combination. In fact, the modem era of clinical transplantation was ushered in by the realization (illustrated in Figure 1) that azathioprine and prednisone can advantageously be used together with an effect exceeding the simple sum of the individual drugs. This combination of agents has been referr-ed to as the « double drug regimen. » THE TRIPLE DRUG REGIMEN OF AZATHIOPRINE, PREDNISONE, AND ALG
A third potent immunosuppressive agent that has been widely employed is heterologous antilymphocyte globulin (ALG). When given subcutaneously, intramuscularly, or intravenously, it has been shown as the sole treatment to mitigate or prevent rejection. Moreover, it has an additive effect when administered with either azathioprine or prednisone. In our clinics, ALG has been administered as a third agent, added to azathioprine and prednisone, in almost all organ recipients treated from May, 1966 to the end of 1970. In most cases the course of ALG therapy was limited to the first 4 post-operative months, during that critical time 37
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120
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Putnam et al.
100
80 60 40
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20
':::cr (ml/min)
BUN (mgm%)-
AZATHIOPRINE ("'Ilm/dayl PREDNISONE (mgm/day)
ALG
=:3
TIME IN DAYS
Fig. 2. -
The course of a patient who received antilymphocyte globulin (ALG) before and for the first four months after renal homotransplantation. The donor was an older brother. There was no early rejection. Prednisone therapy was started 40 days postoperatively because of the high rises in the serologic titers which indicated a host response against the injected foreign protein and which warned against a possible anaphylactic reaction: Note the insidious onset of late rejection after cessation of globulin therapy. Tliis was treated by increasing the maintenance dose of steroids. (By permission of Surg. Gynec. Obstet. 126: 1023, 1968).
when « graft acceptance» is hoped for (Figure 2). We have referred to this treatment program as a triple drug regimen as opposed to a double drug regimen consisting of azathioprine and prednisone only that was used before 1966. . With the institution of the foregoing triple drug program there has been an improvement in the results after renal transplantation at our center. Other workers including those in Lyon, Minneapolis, Salt Lake City, Sydney and Boston have shared this same opinion of the benefits of adding ALG, although it must be conceded that acceptance of ALG as an important comF'onent of treatment has not yet been universal.' This question is an interesting and unresolved one but we will not be able to dwell on it since it is outside the main focus of the present communication. CYCLOPHOSPHAMIDE
With both the double drug and triple drug programs just described, azathioprine has been considered the cornerstone agent. As such, azathioprine has acquired the mystique that goes with presumed indispensibility. The present report will show that another well-known 38
-------------------------------------_._---
Cyclophosphamide and human renal transplantation cytotoxic drug, namely cyclophosphamide (or Cytoxan) is approximately as potent and safe as azathioprine. At the least, it can be administered interchangeably with azathioprine, but hopefully its use may lead to something more than just the advantage of having a major « back-up» drug. As many of you know; azathioprine is a purine analogue. Its site of action has been described by Elion and Hitchings to be principally at the DNA synthesis (or S) phase of the cell cycle (Figure 3). The presumption is that
AZA'fHlIOlPRlINlE
~
"S"
CYClLOlPHO§lPHAMlIDlE
~
"G I " (Interphase)
Fig. 3. Sites of action during the cell cycle of the cytotoxic drugs, azathioprine and cyclophosphamide. Rapidly replicating lymphoid cells are presumably preferentially susceptible to destruction by either agent.
the metabolically active lymphoid tissue which subserves cell-mediated rejection is thus injured and thereby prevented from m.ounting an effective rejection. In contrast, cyclophosphamide is classified as an alkylating agent, chemically similar to nitrogen mustard. Its site of action, according to Wheeler, is at the G2 (or resting)phase of the ccll cycle, just preceding mitosis (Figure 3). Cyclophosphamide in the Triple Drug Regimen Patient and Kidney Mortality Although cyclophosphamide is fundamentally different from azathioprine it has proved capable of performing precisely the same role as exemplified in Figure 4. The patient whose early course is depicted received a kidney more than seven months ago from a mismatched cadaveric donor. Cyclophosphamide was given instead of azathioprine but in doses that were less than half of what would 39
c. W.
100
BUN (mgllOOml)
Cer (ml/min)
WBC (per mm 3 )
RENAL
Putman et al.
TRANSPLANT
80 60 40 20 0++------------------
120~ 100 80 60 40 20 0++-----------------30.000
~ggggW::....-~::::::::::.:::::::=::::~::::::::::::
CYCLOPHOSPHAMIDE ~gg (mg/doy)
PREDNISONE (mg/doy)
ALG
100 50
18gH-------:=~~==3
150 200 DAILY
EV AY
TWICE WEEKLY
01020304050607080
TIME IN DAYS
Fig. 4. - The uncomplicated early convalescence of a cadaveric renal homograft recipient treated with cyclophosphamide, prednisone, and ALG. This patient did not experience a rejection episode. (By permission of Lancet 2 : 70, 1971).
have been anticipated with azathioprine. Rejection in this case was never diagnosed despite the rapid withdrawal of prednisone therapy. ALG was also given initially and later stopped. Figure 5 illustrates the course of another recipient, this time of a maternal kidney. A week after operation there was a severe rejection crisis of the kind first observed under azathioprine therapy almost a decade ago. The findings included recurrent azotemia, a fall in creatinine clearance, oliguria, proteinuria, and hypertension. The crisis was controlled and reversed by temporarily increasing the prednisone dosage. The patient was never given enough cyclophosphamide to depress her white count. This we believe to be the secret of successful cyclophosphamide therapy. In this case, the eventual daily doses were 25 mg or less, or smaller than a half a milligram per kilogram per day. Six to 11 months ago (February to July, 1971) we treated 18 consecutive intrafamilial kidney recipients with the cyclophospha!Uide-containing triple drug regimen. Two of the kidneys underwent hyperacute rejection and were removed immediately or within a few hours; these patients subsequently died after retransplantation. Two other recipients died of perforated diverticulitis and pneumonitis, respectively, for a total patient mortality of 22 %. The other 14 homografts are all functioning satisfactorily (Table 1). In the 3 months from December, 1970 to March, 1971, a comparable group of 20 intrafamilial recipients were treated with the standard triple 40
------_.
-------_.""--"--
Cyclophosphamide and human renal transplantation REJECTION CRISIS
REJECTION
CRISIS
200
WEIGHT 49- 51 KG
14 YEAR OLD 9'
IBO 160 140 120
URI~
BUN
OUTPUT
(mg%)
(mllday)
100 BO 60 40 20 0
so 60
wee
ee,
Imm3 )
(ml/min.)
40
20
0 2.0 URINE
CYCLOPHOSPHAMIDE
PROTEIN (l1m/doy)
(mQ/doy)
1.5 1-0
0 40 39
PREDNISONE
TEMP.
(mQ/doy)
(-C)
38 37
.
ALG
-5
~
t 40 DISCHARGE
TRANSPLANT
DISCHARGE
TIME
60
IN DAYS
Fig. 5. -
The first 60 days after the transplantation of a kidney from a mother to her daughter. Allthough the rejection crisis after a week was a severe one, it was easily and completely reversed. Note that leukopenia was never produced by the daily doses of cyclophosphamide that were usually between 0.5 to 1.0 mg per kg/ day. ALG - horse antilymphocyte globulin; BUN - blood urea nitrogen; CCr - creatinine clearance; WBC - white blood cell count; ARROW - 625 mg methyl prednisolone intravenously. (By permission of Surg. Gynec. Obstet. 133: 981, 1971).
Tahie I. -
Intrafamilial transplantation.
Functioning Grafts* Hyperacute Rejection Patient Dead*
18 Cyclophosphamide
20 Azathioprine
14 (78 %) 2 (11 %)** 4 (22 %)
16 (80 %) 1 (5 %) 3 (15 %)
* Follow-ups in the cyclophosphamide cases are 6 to 1 in the azathioprine cases are 11 to 15 months. ** Both these patients died.
r
months; the follow-ups
drug program containing azathioprine. The results were essentially the same (Table 1). In addition to these intrafamilial cases, 17 cadaveric renal transplantations were also performed under cyclophosphamide. Comparisons with azathioprine were difficult here since the cases were more 41
C. W. Putnam et al. complicated and included numerous retransplantations. In fact, 46 % of the recipients (12 of 26) were undergoing retransplantation for the second to the fifth time. In the cyclophosphamide group the kidney survival to date has been about two-thirds, somewhat better than in the azathioprine controls (Table 2). Tahle 2. -
Cadaveric transplantation* 17 Cyclophosphamide**
Functioning Grafts Hyperacute Rejection Graft Rejected Patient Dead
11 (65 %) 1 2 3 (18 %)
9 A zathioprine**
5 (56 %) 1 2 2 (22 %)
* 12 of these 26 recipients were undergoing retransplantation from the second to the fifth times, helping to explain the relatively poor results in both groups. ** Follow-ups in the cyclophosphamide cases are 6 to 11 months; the follow-ups in the azathioprine cases are 11 to 15 months.
30
~ CYCLOPHOSPHAMIDE
o
(33 PATIENTS) AZATHIOPRINE (29 PATIENTS)
tz 20
w u a::: w
a..
10
o
Fig. 6. -
LEUKOPENIA SERIOUS ANOREXIA ALOPECIA MAJOR MINOR INFECTION OR HEPATIC HEPATIC DIARRHEA DYSFUNCTION DYSFUNCTION
Incidence of toxicity in the first 2 months of treatment of renal homograft recipients with cyclophosphamide versus azathioprine. At the'· doses used, tbere was little obvious difference between the 2 agents. The one patient under cyclophosphamide therapy who developed major hepatic dysfunction had a proven attack of acute serum hepatitis, Australia antigen positive. (By permission of Surg. Gynec. Obstet. 133: 981, 1971).
42
Cyclophosphamide and human renal transplantation Relative Toxicity - As with so many other really important things in organ transplantation, Dr. Will Goodwin of Los Angeles pioneered the use of cydophosphamide almost a decade ago. Unfortunately, in these early days of its use in transplantation and for cancer chemotherapy, cyclophosphamide acquired the reputation of being terribly toxic. We now believe that this bad name was the product of overdosage. In the doses used for our patients, the incidence of serious infections and bone marrow depression were not different than in control cases using azathioprine as shown in Figure 6. The relatively specific complications of cyclophosphamide, such as gastrointestinal complaints, alopecia and hemorrhagic cystitis were either not common or were not observed at all. To give some idea of relatively safe doses of cyclophosphamide in relation to those of azathioprine, the statistical study depicted in Figure 7 was
~
o
C'I'CLOPrKlSPHAMIDE AZATHIOPRINE
(l6 PATIENTS)
(19 PATIENTS)
l ih
CYCLOPHOSPHAMIDE OR WHITE BLOOD CELL AZATHIOPRINE COUNT
LYMPHOCYTE COUNT
:-~~ 15'OOO~ ~ li.l 1= F': .. 2
3,000
o
500
o · I MONTH
i MONTH p< 025
2 MONTHS p~
11
